The innate arm of the immune system can critically shape the adaptive immune response against pathogens. Thus, innate sensing mechanisms have been studied extensively for rational adjuvant design. In contrast, much less is known about the immunological impact of 'restriction factor' genes that could directly inhibit virus replication. Tetherin is the protein counteracted by Vpu of pandemic HIV-1 to promote virion release. In the absence of Vpu, Tetherin 'tethers' virions on the cell surface. Subsequently, these tethered virions are brought back into the infected cell through Tetherin-mediated endocytosis. The immunobiology of Tetherin remains unknown, and in vitro studies raised controversies on whether it acts as a retrovirus restriction factor. Thus, in vivo studies that isolate the gene for controlled study may help advance insights and resolve discrepancies on Tetherin function. In addition, investigating the biology of Tetherin in vivo may benefit from disrupting its mysterious endocytic function. Recently, we identified an inbred mouse strain that harbored a Tetherin single nucleotide polymorphism (SNP) that resulted in an endocytosis defect (Barrett et al. PLOS Pathogens 2012). In published and unpublished genetic backcrossing studies, we discovered that the impact of this Tetherin endocytosis SNP on Friend retrovirus (FV) infection is dependent on the Major Histocompatibility Complex (MHC) locus. Moreover, MHC Class II+ antigen-presenting cells (APCs) expressing Tetherin stimulated an FV- specific CD4+ T cell hybridoma to a greater extent than Tetherin-deficient APCs. Thus, Tetherin-mediated virion endocytosis in APCs may increase the viral peptide pool for MHC Class II presentation and augment the retrovirus-specific CD4+ T cell response. We propose to test this hypothesis using two complementary systems. In Specific Aim 1, we will evaluate the ability of APCs expressing wild-type, knock- out and endocytosis-defective Tetherin to stimulate CD4+ T cells in the FV infection model. Since Tetherin may also impact cross-presentation to MHC Class I, we will also investigate the CD8+ T cell response. In Specific Aim 2, we will test if enabling Tetherin activity through a disruptive Vpu mutation would render the HIV-1 infected target cells more susceptible to the cytolytic and suppressive effects of HIV-1 specific CD4+ T cell clones in vitro. Concordant results from the proposed in vivo immunologic studies and HIV-1 in vitro studies may provide insights in harnessing Tetherin biology for retroviral immunotherapeutics and T-cell vaccines.